1. Field of the Invention
This invention relates to the field of molecular biology to create genetically modified bacteria resistant to and capable of sequestering and accumulating heavy metals, including mercury, lead, zinc, and cadmium, for bioremediation of contaminated liquids and solids.
2. Description of the Background
Metallic chemical elements that have a relatively high density are often referred to as heavy metals. The heavy metals are toxic even at low concentrations. Toxic heavy metals include mercury, cadmium, lead, zinc and silver. Among the heavy metals, mercury, lead, and cadmium are considered particularly toxic.
Mercury has been introduced into the environment as a byproduct of industrial and natural processes and can accumulate in soil and sediments in high concentrations. Patra, M. and Sharma A., Bot. Rev. 66:379-422 (2000). In the United States, coal burning power plants emit about 48 tons of mercury annually, while in Asia and Africa coal burning power plants release more than 1500 tons per year. Clean Air Mercury Rule. U.S. Environmental Protection Agency (“EPA”) 2009, Retrieved Feb. 8, 2009 from the U.S. Environmental Protection Agency website. Globally the annual mercury emissions from all sources are estimated at 4800-8300 tons. Mercury Human Exposure, EPA 2008, Retrieved Feb. 8, 2009 from the U.S. Environmental Protection Agency website.
Mercury compounds are neurotoxins and potent blockers of electron transport in the cell. All mercury forms are toxic and present risks to human health and to the environment. Developing a cost-efficient and effective remediation system is of utmost importance.
Current remediation strategies to clean mercury from the environment include flushing, chemical reduction/oxidation, excavation, retrieval and off-site disposal. These approaches are expensive, environmentally disruptive, and inefficient. Karenlampi, S. et al., Environ. Pollut. 1007:225-231 (2000). Other methods, such as vitrification and concrete capping, render the site unusable and are impractical in remediation of large areas. The cost of remediating a pound of mercury from the environment with current technologies is in the several thousands of dollars. Hussein, H. et al., Env. Sci. Technol. 41:8439-8446 (2007).
Like mercury, other heavy metals, such as lead and cadmium, present a serious environmental threat and must be remediated. Lead is a powerful neurotoxin that can accumulate in soft tissue and bones. Because of its toxicity lead has been banned by the EPA and other Agencies from consumer products including paints, gasoline, water pipes, toys, and others. The EPA limits lead content to less than 0.015 ppm in drinking water. Lead ranks second in the 2007 Comprehensive Environmental Response Compensation, and Liability Act (CERCLA) priority list of hazardous substances.
The current wide spread use of cadmium in multiple consumer applications, especially in batteries, has increased environmental pollution of this heavy metal. Cadmium has been shown to be highly toxic, causing serious poisoning, bone degeneration, cellular enzymes inhibition, and cell membrane disruption. As in the case of mercury, current methods to remediate or capture lead and cadmium rely on the use of physicochemical methods including the use of ion exchange resins, precipitation and extraction, burial, site capping, and offsite disposal. Kim, S. et al. J. Biosci. Bioeng. 99:109-114 (2005). These methods are costly and/or disruptive to the environment being reclaimed. New technologies are required to facilitate the remediation of contaminated environments.
Bioremediation, the use of organisms for the restoration of contaminated environments, may present a potentially low cost and environmentally friendly approach. For example, bacteria can break down certain toxic compounds into their non-toxic metabolites. However, heavy metal elements, such as mercury, cadmium and lead, can not be detoxified into non-toxic metabolites.
A method of mercury bioremediation, by volatilization of mercury, relies on the expression of the mer operon, which manages the transport and reduction of Hg2+. One of the mer operon genes, merA, codes for mercuric ion reductase, an enzyme that catalyzes the conversion of Hg2+ to Hg0. Hg0 is a less volatile, less-reactive and less toxic form of mercury. Jackson, W. J. and Summers, A. O., J. Bacteriol. 151:962-970 (1982). In the volatilization process, however, elemental mercury is released into the environment where it can be converted into more toxic forms. Another disadvantage to the volatilization method is that it is not suitable for water treatment, because bacteria release the volatilized elemental mercury into the same water that is being remediated.
Bacteria do not have endogenous mechanisms that provide high resistance to mercury, while allowing mercury accumulation inside the cell. Genetic engineering has been used to integrate genes from other organisms with the goal of increasing mercury resistance and accumulation. Molecules known as chelators or sequestration agents have been proposed as suitable heavy metal scavenging agents that can be expressed in organisms with the goal of recovering the heavy metals from soil or water.
Metallothionein and polyphosphate in bacterial systems have been implied in the detoxification of some heavy metals. These two agents, expressed in E. coli, can sequester mercury, cadmium and lead and thus protect the bacteria from certain levels of these heavy metal elements. The results to date, however, have been discouraging. The bacteria can not effectively sequester these elements and do not survive high levels of these heavy metals. These results are attributed to a perceived lack of stability of the chelator protein agent, creating bacterial systems with weak tolerance for the heavy metal.
Metallothioneins are encoded by the mt genes found in mammals, plants, and fungi. Sousa, C. et al., J. Bacteriol. 180:2280-2284 (1998). Metallothionein (MT), however, has been shown to be unstable when expressed in bacteria. Berka, T. et al., J. Bacteriol. 170:21-26 (1988). Because the MT protein was found to be unstable when expressed in bacteria, the mt gene has been fused with stabilizing agents such as glutathione-5-transferase (GST) creating GST-MT fusions. Chen, S. and Wilson D. B., Appl. Env. Microbiol. 63:2442-2445 (1997). Various GST-MT constructs included S. cerevisiae (GST-YMT), human (GST-HMT) and pea (GST-PMT). Cells harboring GST-HMT have not been shown to produce soluble MT proteins and the construct does not confer any resistance to mercury. Cells expressing the YMT and PMT constructs have been shown to tolerate liquids having at most 5 μM mercury, a level that is barely toxic. More importantly, cells expressing these two constructs do not appear to accumulate mercury or protect the cell from mercury, unless the cell is further engineered to express mercury transport genes of the mer operon. Various unsuccessful attempts have also been made to engineer multiple copies of mt gene of N. crassa and other human mt genes, targeted to the bacterial periplasm. The instability and insolubility of these proteins, however, have continued to prevent their use as effective remediation agents. Valls, M. and Lorenzo, V., FEMS Micro. Reviews, 26:327-338 (2002). Although these fusions proteins confer some limited tolerance to mercury, this effect can not be clearly attributed to the MT proteins because GST, the fusion partner, is also known to bind heavy metals such as mercury. Chen, S. and Wilson D. B., Appl. Environ. Microbiol. 63:2442-2445 (1997); Deng, X. and Wilson D. B., Appl. Microbiol. Biotechnol. 56:276-279 (2001); Custodio, H. M., et al., Arch. Environ. Occup. Health 60:17-23 (2005).
Therefore, it has been concluded that the transgenic bacteria modified with metallothionein genes have not provided adequate resistance in cells. Beattie, J. H. et al., Toxicol. Lett. 157:69-78 (2005); Odawara, F. et al., J. Biochem. 118:1131-1137 (1995); Park, J. D., et al., Toxicology. 163:93-100 (2001). Explanations given for this failure include rapid degradation of the small metallothionein peptide by cellular proteases, low protein yield, and possible interference with redox pathways in the cytosol. Sousa, C. et al., J. Bacteriol. 180:2280-2284 (1998); Yang, F. et al., Protein Expr. Purif. 53:186-194 (2007).
Also, attempts to engineer bacteria with metallothionein genes to enhance resistance to zinc have proven ineffective. Odawara, F. et. al., supra.
Metallothionein fusion genes expressed in bacteria have shown to provide but marginal tolerance to cadmium toxicity to up to 50 mg/liter (about 150 μM). Odawara, F. et. al., Id.; Keasling, J. D., and Hupf, G. A. Applied Env. Microbiol. 62:743-746 (1996). These studies do not indicate that bacteria can grow well in high cadmium concentrations because after 50 mg/L of cadmium the transgenic cell had a substantial decrease in growth in comparison with transgenic cells growing in media without cadmium.
Others have focused on engineering the polyphosphate kinase (“ppk”) gene for expression in bacteria. The ppk enzyme is responsible for the synthesis of long linear polymers of orthophosphates known to absorb (sequester) mercury. Similarly to mt, only ppk fusion constructs have been proposed and utilized. For example, the Klebsiella aerogenes ppk gene has been fused with Pseudomonas derived merT and merP genes. The merT and merP genes facilitate internalization of mercury. The fusion was meant to improve stability and the fusion components were chosen in part due to the belief that mercury internalization would be limited, which would also limit the bioremediation effect of the bacteria expressing the ppk gene. Pan-Hou, H. et al., Biol. Pharm. Bull. 24:1423-1426 (2001); Pan-Hou, H. et al., FEMS Microbiol. Lett. 10325:159-164 (2002). Bacteria expressing these constructs are capable of accumulating up to 16 μM mercury and 24 μM of an organo-mercury compound from solutions. Bacterial growth in the presence of elemental mercury was abolished at 16 μM mercury. Increased resistance to mercury has been shown when the engineered bacteria expressing the constructs were placed on alginate beads. Nevertheless, mercury remediation is inactivated and the bacteria loses viability in the presence of 40-80 μM mercury. Kyono, M. et al., Appl. Microbiol. Biotechnol. 62:274-278 (2003).
Phytoremediation and mycoremediation (non-engineered organisms) have been the methods used to attempt to bioremediate lead, by accumulating lead in the roots or leaf. Huang, L. Z. et al., Biodegradation 20:651-660 (2009); Vimala, R. and Das, N., J., Hazard. Mat. 168:376-382 (2009). No effective bacterial bioremediation technology has been proposed for lead as of today.
The low level of resistance of the engineered bacteria to the heavy metal achieved by the above mentioned systems preclude their application as an effective bioremediation system. Even in water with low mercury concentrations, these systems would not be effective because mercury will accumulate in the cell to concentrations higher than what is tolerated by the system.